NCBI Non-redundant Protein Research Articles

Generation and analysis of expressed sequence tags from adductor muscle of Japanese scallop Mizuhopecten yessoensis

A normalized cDNA library was constructed from the adductor muscle of M. yessoensis and acquired 4595 high quality expressed sequence tags (ESTs). After clustering and assembly of the ESTs, 3061 unigenes containing 654 contigs and 2407 singletons were identified. The contig length ranged from 266 bp to 2364 bp and the average length of these contigs was 544 bp. Blastx nonredundant protein database analysis showed that 1522 unigenes had significant homology to known genes (E value ≤ 10 − 5 ). By comparing to Clusters of Orthologous Groups (COG) categories, 460 unigenes were annotated (E value ≤ 10 − 10 ). Using Kyoto Encyclopedia of Genes and Genomes (KEGG), 345 of 3061 unigenes were assigned into 103 pathways (E value ≤ 10 − 5 ). For InterProScan searches, 1237 unigenes were annotated containing 727 different types of protein domains. 941 of the 1237 unigenes were annotated for Gene Ontology (GO) classification using Uniprot2GO associations in any category (biological, cellular, and molecular). By sequences comparability and analysis of Blastx NCBI nonredundant protein database and KEGG, 66 unigenes were identified that may be involved in genetic information processing based on the known knowledge. The study provides a material basis as useful information for the genomic analysis of shellfish.

Genes differentially expressed by Aspergillus carbonarius strains under ochratoxin A producing conditions

Aspergillus carbonarius is an important ochratoxin A (OTA)-producing fungus that is responsible for toxin contamination of grapes and wine, coffee and cocoa. A suppression subtractive hybridization (SSH) approach was performed with two strains of A. carbonarius, antagonistic in their OTA-production ability, to identify genes whose expression is linked with the ability to produce OTA. BlastX analysis identified 109 differentially-expressed sequences putatively involved in the production of OTA, with significant similarities ( E value < 10 − 5 ) to sequences deposited in the NCBI non-redundant protein database. Of the 109 ESTs, 26% were involved in regulation processes, 15% corresponded to hypothetical proteins, 12% were involved in stress response and detoxification, 9% corresponded to transport and secretion processes, 7% corresponded to amino acid metabolism, 7% were involved in hydrolysis of energy reserves and 5% involved in secondary metabolism. Other unisequences showed homology to genes involved in protein synthesis and general metabolism. According to their sequence similarities to genes in the NCBI database, the possible functional roles they might play in the production and regulation of OTA are discussed. Worth noting is the high percentage of genes involved in regulation, including specific and global regulators. It is also important to note the high percentage of genes involved in the response to stress and detoxification.

Proteomic analysis of pollination-induced corolla senescence in petunia

Senescence represents the last phase of petal development during which macromolecules and organelles are degraded and nutrients are recycled to developing tissues. To understand better the post-transcriptional changes regulating petal senescence, a proteomic approach was used to profile protein changes during the senescence of Petunia×hybrida ‘Mitchell Diploid’ corollas. Total soluble proteins were extracted from unpollinated petunia corollas at 0, 24, 48, and 72 h after flower opening and at 24, 48, and 72 h after pollination. Two-dimensional gel electrophoresis (2-DE) was used to identify proteins that were differentially expressed in non-senescing (unpollinated) and senescing (pollinated) corollas, and image analysis was used to determine which proteins were up- or down-regulated by the experimentally determined cut-off of 2.1-fold for P <0.05. One hundred and thirty-three differentially expressed protein spots were selected for sequencing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the identity of these proteins. Searching translated EST databases and the NCBI non-redundant protein database, it was possible to assign a putative identification to greater than 90% of these proteins. Many of the senescence up-regulated proteins were putatively involved in defence and stress responses or macromolecule catabolism. Some proteins, not previously characterized during flower senescence, were identified, including an orthologue of the tomato abscisic acid stress ripening protein 4 (ASR4). Gene expression patterns did not always correlate with protein expression, confirming that both proteomic and genomic approaches will be required to obtain a detailed understanding of the regulation of petal senescence.

Global characterization of Artemisia annua glandular trichome transcriptome using 454 pyrosequencing

BackgroundGlandular trichomes produce a wide variety of commercially important secondary metabolites in many plant species. The most prominent anti-malarial drug artemisinin, a sesquiterpene lactone, is produced in glandular trichomes of Artemisia annua. However, only limited genomic information is currently available in this non-model plant species.ResultsWe present a global characterization of A. annua glandular trichome transcriptome using 454 pyrosequencing. Sequencing runs using two normalized cDNA collections from glandular trichomes yielded 406,044 expressed sequence tags (average length = 210 nucleotides), which assembled into 42,678 contigs and 147,699 singletons. Performing a second sequencing run only increased the number of genes identified by ~30%, indicating that massively parallel pyrosequencing provides deep coverage of the A. annua trichome transcriptome. By BLAST search against the NCBI non-redundant protein database, putative functions were assigned to over 28,573 unigenes, including previously undescribed enzymes likely involved in sesquiterpene biosynthesis. Comparison with ESTs derived from trichome collections of other plant species revealed expressed genes in common functional categories across different plant species. RT-PCR analysis confirmed the expression of selected unigenes and novel transcripts in A. annua glandular trichomes.ConclusionThe presence of contigs corresponding to enzymes for terpenoids and flavonoids biosynthesis suggests important metabolic activity in A. annua glandular trichomes. Our comprehensive survey of genes expressed in glandular trichome will facilitate new gene discovery and shed light on the regulatory mechanism of artemisinin metabolism and trichome function in A. annua.

Identification and Characterization of a Novel Nuclear Protein Complex Involved in Nuclear Hormone Receptor-mediated Gene Regulation

NRC/NCoA6 plays an important role in mediating the effects of ligand-bound nuclear hormone receptors as well as other transcription factors. NRC interacting factor 1 (NIF-1) was cloned as a novel factor that interacts in vivo with NRC. Although NIF-1 does not directly interact with nuclear hormone receptors, it enhances activation by nuclear hormone receptors presumably through its interaction with NRC. To further understand the cellular and biological function of NIF-1, we identified NIF-1-associated proteins by in-solution proteolysis followed by mass spectrometry. The identified components revealed factors involved in histone methylation and cell cycle control and include Ash2L, RbBP5, WDR5, HCF-1, DBC-1, and EMSY. Although the NIF-1 complex contains Ash2L, RbBP5, and WDR5, suggesting that the complex might methylate histone H3-Lys-4, we found that the complex contains a H3 methyltransferase activity that modifies a residue other than H3-Lys-4. The identified components form at least two distinctly sized NIF-1 complexes. DBC-1 and EMSY were identified as integral components of an NIF-1 complex of approximately 1.5 MDa and were found to play an important role in the regulation of nuclear receptor-mediated transcription. Stimulation of the Sox9 and HoxA1 genes by retinoic acid receptor-alpha was found to require both DBC-1 and EMSY in addition to NIF-1 for maximal transcriptional activation. Interestingly, NRC was not identified as a component of the NIF-1 complex, suggesting that NIF-1 and NRC do not exist as stable in vitro purified complexes, although the separate NIF-1 and NRC complexes appear to functionally interact in the cell.

Transcriptome analysis of the central nervous system of the mollusc Lymnaea stagnalis

BackgroundThe freshwater snail Lymnaea stagnalis (L. stagnalis) has served as a successful model for studies in the field of Neuroscience. However, a serious drawback in the molecular analysis of the nervous system of L. stagnalis has been the lack of large-scale genomic or neuronal transcriptome information, thereby limiting the use of this unique model.ResultsIn this study, we report 7,712 distinct EST sequences (median length: 847 nucleotides) of a normalized L. stagnalis central nervous system (CNS) cDNA library, resulting in the largest collection of L. stagnalis neuronal transcriptome data currently available. Approximately 42% of the cDNAs can be translated into more than 100 consecutive amino acids, indicating the high quality of the library. The annotated sequences contribute 12% of the predicted transcriptome size of 20,000. Surprisingly, approximately 37% of the L. stagnalis sequences only have a tBLASTx hit in the EST library of another snail species Aplysia californica (A. californica) even using a low stringency e-value cutoff at 0.01. Using the same cutoff, approximately 67% of the cDNAs have a BLAST hit in the NCBI non-redundant protein and nucleotide sequence databases (nr and nt), suggesting that one third of the sequences may be unique to L. stagnalis. Finally, using the same cutoff (0.01), more than half of the cDNA sequences (54%) do not have a hit in nematode, fruitfly or human genome data, suggesting that the L. stagnalis transcriptome is significantly different from these species as well. The cDNA sequences are enriched in the following gene ontology functional categories: protein binding, hydrolase, transferase, and catalytic enzymes.ConclusionThis study provides novel molecular insights into the transcriptome of an important molluscan model organism. Our findings will contribute to functional analyses in neurobiology, and comparative evolutionary biology. The L. stagnalis CNS EST database is available at .

Tyrosylprotein Sulfotransferase-2 Expression Is Required for Sulfation of RNase 9 and Mfge8 in Vivo

Protein-tyrosine sulfation is mediated by two Golgi tyrosyl-protein sulfotransferases (TPST-1 and TPST-2) that are widely expressed in vivo. However, the full substrate repertoire of this enzyme system is unknown and thus, our understanding of the biological role(s) of tyrosine sulfation is limited. We reported that whereas Tpst1(-/-) male mice have normal fertility, Tpst2(-/-) males are infertile despite normal spermatogenesis. However, Tpst2(-/-) sperm are severely defective in their motility in viscous media and in their ability to fertilize eggs. These findings suggest that sulfation of unidentified substrate(s) is crucial for normal sperm function. We therefore sought to identify tyrosine-sulfated proteins in the male genital tract using affinity chromatography on PSG2, an anti-sulfotyrosine monoclonal antibody, followed by mass spectrometry. Among the several candidate tyrosine-sulfated proteins identified, RNase 9 and Mfge8 were examined in detail. RNase 9, a catalytically inactive RNase A family member of unknown function, is expressed only in the epididymis after onset of sexual maturity. Mfge8 is expressed on mouse sperm and Mfge8(-/-) male mice are subfertile. Metabolic labeling coupled with sulfoamino acid analysis confirmed that both proteins are tyrosine-sulfated and both proteins are expressed at comparable levels in wild type, Tpst1(-/-), and Tpst2(-/-) epididymides. However, we demonstrate that RNase 9 and Mfge8 are tyrosine-sulfated in wild type and Tpst1(-/-), but not in Tpst2(-/-) mice. These findings suggest that lack of sulfation of one or both of these proteins may contribute mechanistically to the infertility of Tpst2(-/-) males.

Different proteomic protein patterns in saliva of Sjögren's syndrome patients

To investigate the salivary protein profile in patients with Sjögren's syndrome (SS), and healthy control subjects. Unstimulated whole saliva samples were collected from 16 age-matched females; eight healthy subjects and eight patients diagnosed with SS (six primary SS, one incomplete SS and one primary SS associated with B cell lymphoma). Proteins were extracted and separated individually by 2D sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Selected protein spots of interest were analysed by electrospray ionization--tandem mass spectrometry. Obtained data were searched against the Swiss-Prot and NCBI non-redundant protein databases using Mascot software. Two groups of patterns of protein expression were observed in the eight SS patients: a major group (six patients) with significant expression differences from the healthy subjects and the second group (two patients) with a pattern similar to the eight healthy subjects. In this preliminary study, protein expression differences were found between SS patients and healthy subjects. Individual analysis of SS patients exhibited two patterns of protein expression with no direct relation to the clinical, serological or histological severity of disease. This study emphasizes the difficulty of the present proteomic knowledge to diagnose and monitor the sequel of SS development.

Laser capture microdissection and expressed sequence tag analysis of uredinia formed by Phakopsora pachyrhizi, the causal agent of Asian soybean rust

Soybean is one of the top five agricultural products in the United States. Soybean rust is caused by the obligate fungus Phakopsora pachyrhizi Sydow, an exotic pathogen in the U.S. Extensive screening of soybean germplasm has not identified soybean with resistance to all of the different isolates of Asian soybean rust. A biotechnological approach may help to broaden resistance of soybean to this fungus. Using laser capture microdissection, we isolated uredinia formed by P. pachyrhizi on the under side of leaves, extracted the RNA and constructed a cDNA library. A total of 925 cDNAs were sequenced from the library, contigs were formed, and homology searches were conducted to determine the identity of the genes. Forty-one unisequences with significant similarities ( e-value ≤ 10 −5) to sequences deposited in the NCBI non-redundant protein databases were identified. Sixteen had similarities to known genes encoding proteins involved in different cellular functions such as energy production, cellular communication/signal transduction, and transcription; twenty-four represented genes encoding hypothetical proteins or proteins with unknown function. Ten unisequences of unknown protein function included conserved domains from InterPro databases. Of the 41 unisequences, 61% were similar to fungal sequences. RT-PCR was used to explore the expression pattern of seventeen rust genes, revealing some targeted for additional study of control of P. pachyrhizi in soybean.

Proteomic profile of dormant Trichophyton Rubrum conidia

BackgroundTrichophyton rubrum is the most common dermatophyte causing fungal skin infections in humans. Asexual sporulation is an important means of propagation for T. rubrum, and conidia produced by this way are thought to be the primary cause of human infections. Despite their importance in pathogenesis, the conidia of T. rubrum remain understudied. We intend to intensively investigate the proteome of dormant T. rubrum conidia to characterize its molecular and cellular features and to enhance the development of novel therapeutic strategies.ResultsThe proteome of T. rubrum conidia was analyzed by combining shotgun proteomics with sample prefractionation and multiple enzyme digestion. In total, 1026 proteins were identified. All identified proteins were compared to those in the NCBI non-redundant protein database, the eukaryotic orthologous groups database, and the gene ontology database to obtain functional annotation information. Functional classification revealed that the identified proteins covered nearly all major biological processes. Some proteins were spore specific and related to the survival and dispersal of T. rubrum conidia, and many proteins were important to conidial germination and response to environmental conditions.ConclusionOur results suggest that the proteome of T. rubrum conidia is considerably complex, and that the maintenance of conidial dormancy is an intricate and elaborate process. This data set provides the first global framework for the dormant T. rubrum conidia proteome and is a stepping stone on the way to further study of the molecular mechanisms of T. rubrum conidial germination and the maintenance of conidial dormancy.

Identification of soluble secreted proteins from appressoria of Colletotrichum higginsianum by analysis of expressed sequence tags

The hemibiotrophic ascomycete Colletotrichum higginsianum causes anthracnose disease on brassica crops and the model plant Arabidopsis. Melanized appressoria pierce the host cuticle and cell wall to form specialized biotrophic hyphae inside living epidermal cells. To identify proteins secreted by appressoria that may function as virulence effectors, a cDNA library was prepared from mature appressoria formed in vitro. Bidirectional sequencing of 980 clones generated 1442 high-quality expressed sequence tags (ESTs), comprising 518 unique sequences. blastx analysis showed that 353 (68 %) of these had significant similarity to entries in the NCBI non-redundant protein database, of which 49 were also homologous to experimentally verified fungal pathogenicity genes. ORFs were predicted ab initio from the unique sequences and screened for potential signal peptides using SignalP. Fifty-three unique sequences (10 %) were predicted to encode proteins entering the secretory pathway, of which 26 were likely to be soluble secreted proteins. For a selected subset of these, RT-PCR showed that seven genes that encode secreted proteins of unknown function, including two Colletotrichum-specific genes, are upregulated in appressoria and expressed early during plant infection, and therefore represent candidate effectors.

Purification and characterization of three novel keratinolytic metalloproteases produced by Chryseobacterium indologenes TKU014 in a shrimp shell powder medium

A protease-producing bacterium was isolated and identified as Chryseobacterium indologenes TKU014. The optimized condition for protease production was found when the culture was shaken at 30 °C for one day in 50 mL of medium containing 0.5% shrimp shell powder (w/v), 0.1% K 2HPO 4, and 0.05% MgSO 4 · 7H 2O. Three extracellular proteases (P1, P2, and P3) were purified from culture by DEAE-Sepharose and Phenyl Sepharose chromatography. Three enzymes all showed activities of keratinase and elastase with molecular weights of 56, 40, 40 kDa, respectively. The inhibitory effect of metal chelator EDTA and Zn-specific chelator 1,10-phenanthroline characterized three enzymes as Zn-metalloproteases. Peptide mass fingerprints of P1, P2, and P3 were determined by using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Similarity search in the NCBI non-redundant protein sequence database revealed that three enzymes exhibited no significant homology to any other reported microbial peptides. Therefore, P1, P2, and P3 are most likely novel proteins.

PROTEIN PROFILING OF MOUSE OOCYTES MATURED IN VITRO DURING THE FIRST WAVE OF FOLLICULOGENESIS

During the first month after birth, synchronous follicular growth occurs in the ovaries of immature mice (the first wave). Previously, we showed that mouse oocytes during the first wave were more competent developmentally in older females, although the numbers of mature oocytes did not differ with female age (17, 18, and 24 days old). In this study, we profiled the proteins of oocytes matured in vitro during the first wave using two-dimensional electrophoresis (2-DE) as a first step in search of proteins involved in the maturation of mouse oocytes. Oocytes collected from ovarian follicles of B6D2F1 females at nine ages (from 17 to 25 days of age) without hormonal treatment were matured in Waymouth medium (Sigma) supplemented with pyruvate (0.23 mM), antibiotics, polyvinylpyrrolidone (3 mg mL−1), and recombinant human FSH (0.5 iu mL−1, Serono). After culturing for 17 h at 37°C in an atmosphere of 5% CO2, 5% O2, and 90% N2, oocytes whose germinal vesicles had broken down (mature oocytes) were dissolved in rehydration/sample buffer (8 M urea, 2% CHAPS, 50 mM DTT, 0.2% Bio-Lyte 3/10, and 0.001% BPB; Bio- Rad). In the first dimension, 4–10 μg of proteins from 200–400 mature oocytes were separated by isoelectric focusing using IPG ReadyStrip pH 3– 10 (7 cm in length, Bio-Rad) with a Protean IEF Cell (Bio-Rad). After alkylation with iodoacetamide, the proteins were separated in the second dimension using SDS-PAGE gradient gels (ReadyGel J 10–20%, gel size: 80[W] x 73[L] mm, Bio-Rad). The protein spots on the gels were visualized using SYPRO Ruby stain (Invitrogen). Approximately 200 protein spots were detected on each gel. We identified six commonly detected proteins on 2-DE gels throughout the first wave using peptide mass fingerprinting and a Mascot search against the NCBI nonredundant protein sequence database to identify protein spots that can be used as protein landmarks for future 2-DE analyses. The most prominent proteins were lactate dehydrogenase B (37 kDa, pI=5.70) and ubiquitin carboxy-terminal hydrolase L1 (25.2 kDa, pI=5.33). The other proteins were tumor rejection antigen gp96 (92.7 kDa, pI=4.74), a fragment of zona pellucida glycoprotein 2 (~18 kDa, pI ~4; the amino acid sequence was confirmed by MS/MS analysis), pyruvate kinase M (58.4 kDa, pI=7.58), and peroxiredoxin 1 (22.4 kDa, pI=8.26). Our results provide a basis for further 2-DE analyses of the proteins involved in the maturation of mouse oocytes. This work was supported by a grant from the Ministry of Health, Labor, and Welfare of Japan. (poster)

Large Scale Identification of Proteins, Mucins, and Their O-Glycosylation in the Endocervical Mucus during the Menstrual Cycle

The mucus filling the human cervical opening blocks the entry to the uterus, but this has to be relative and allow for the sperm to penetrate at ovulation. We studied this mucus, its content of proteins and mucins, and the mucin O-glycosylation in cervical secretions before, during, and after ovulation. Cervical mucosal secretions from 12 subjects were collected, reduced-alkylated, separated with polyacrylamide or agarose/polyacrylamide gel electrophoresis, and stained with silver, Alcian blue, or Coomassie Blue stain. Protein and mucin bands from before and during ovulation were digested and subsequently analyzed by nano-LC-FT-ICR MS and MS/MS. We identified 194 proteins after searches against the NCBI non-redundant protein database and an in-house mucin database. Three gel-forming (MUC5B, MUC5AC, and MUC6) and two transmembrane mucins (MUC16 and MUC1) were identified. For the analysis of mucin O-glycosylation, separated mucins from six individuals were blotted to PVDF membranes, and the O-glycans were released by reductive beta-elimination and analyzed with capillary HPLC-MS and -MS/MS. At least 50 neutral, sialic acid-, and sulfate-containing oligosaccharides were found. An increase of GlcNAc-6GalNAcol Core 2 structures and a relative decrease of NeuAc residues are typical for ovulation, and NeuAc-6GalNAcol and NeuAc-3Gal- epitopes are typical for the non-ovulatory phases. The cervical mucus at ovulation is thus characterized by a relative increase in neutral fucosylated oligosaccharides. This comprehensive characterization of the mucus during the menstrual cycle suggests mucin glycosylation as the major alteration at ovulation, but the relation to the altered physicochemical properties and sperm penetrability is still not understood.

Purification and Identification of Proteins That Bind to the Hereditary Persistence of Fetal Hemoglobin –198 Mutation in the γ-Globin Gene Promoter

Expression of the gamma-globin gene is silenced in adult humans. However, certain point mutations in the gamma-globin gene promoter are capable of maintaining expression of this gene during adult erythropoiesis, a condition called non-deletion hereditary persistence of fetal hemoglobin (HPFH). Among these, the British form of HPFH carrying a T-->C point mutation at position -198 of the Agamma-globin gene promoter results in 4-10% fetal hemoglobin in heterozygotes. In this study, we used nuclear extracts from murine erythroleukemia cells to purify a protein complex that binds the HPFH -198 gamma-globin gene promoter. Members of this protein complex were identified by mass spectrometry and include DNMT1, the transcriptional coactivator p52, the protein SNEV, and RAP74 (the largest subunit of the general transcription factor IIF). Sp1, which was previously considered responsible for HPFH -198 gamma-globin gene activation, was not identified. The potential role of these proteins in the reactivation and/or maintenance of gamma-globin gene expression in the adult transcriptional environment is discussed.

Pyrrolidine Dithiocarbamate Inhibits Interleukin-6 Signaling through Impaired STAT3 Activation and Association with Transcriptional Coactivators in Hepatocytes

Pyrrolidine Dithiocarbamate Inhibits Interleukin-6 Signaling through Impaired STAT3 Activation and Association with Transcriptional Coactivators in Hepatocytes

A novel fibrinolytic serine protease from the polychaete Nereis ( Neanthes) virens ( Sars): Purification and characterization

A novel fibrinolytic serine protease has been identified and purified to homogeneity from the coelomic fluid of polychaete Nereis ( Neanthes) virens ( Sars), and named N-V protease. N-V protease is a 29 kDa single chain protein with an isoelectric point of pH 4.5. It hydrolyzes Aα-chain of fibrinogen with a high efficiency, and the Bβ- and γ-chains (Aα > Bβ > γ) with a lower efficiency. The proteolytic activity peaks at pH 7.8 is 45 °C. The activity is completely inhibited by serine protease inhibitors DFP (I 50 = 5.8 × 10 −4 M) and PMSF (I 50 = 5.5 × 10 −2 M), and almost completely by TLCK (I 50 = 7.7 × 10 −1 M). But aprotinin, elastinal, SBTI, benzamidine, PCMB, EDTA, EGTA, iodoacetate, E64, and β-mercaptoethanol have no effect on the protease activity. Therefore, N-V protease is identified as a serine protease. The primary amino acid sequence of N-V protease was determined by mass spectrometry ( N-V protease, No. P83433). According to the MALDI-TOF MS analysis, there is no existing protein in the NCBI Non-redundant Protein Sequence Database that matches the N-V protease sequence. Therefore, N-V protease is a novel and special protein in N. virens. Furthermore, we have successfully established an expression cDNA library from the whole body of N. virens (data not shown).

Proteomic and genetic approaches to identifying defence‐related proteins in rice challenged with the fungal pathogen Rhizoctonia solani

SUMMARY Sheath blight, caused by the fungus Rhizoctonia solani, is a major disease of rice world-wide, but little is known about the host response to infection. The objective of this study was to identify proteins and DNA markers in resistant and susceptible rice associated with response to infection by R. solani. Replicated two-dimensional polyacrylamide gel electrophoresis experiments were conducted to detect proteins differentially expressed under inoculated and non-inoculated conditions. Tandem mass spectra analysis using electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS) was carried out for protein identification with the NCBI non-redundant protein database. Seven proteins were increased after inoculation in both susceptible and resistant plants. Six of the seven proteins were identified with presumed antifungal, photosynthetic and proteolytic activities. An additional 14 proteins were detected in the response of the resistant line. Eleven of the 14 proteins were identified with presumed functions relating to antifungal activity, signal transduction, energy metabolism, photosynthesis, molecular chaperone, proteolysis and antioxidation. The induction of 3-beta-hydroxysteroid dehydrogenase/isomerase was detected for the first time in resistant rice plants after pathogen challenge, suggesting a defensive role of this enzyme in rice against attack by R. solani. The chromosomal locations of four induced proteins were found to be in close physical proximity to genetic markers for sheath blight resistance in two genetic mapping populations. The proteomic and genetic results from this study indicate a complex response of rice to challenge by R. solani that involves simultaneous induction of proteins from multiple defence pathways.

Analysis of 10,000 ESTs from lymphocytes of the cynomolgus monkey to improve our understanding of its immune system

BackgroundThe cynomolgus monkey (Macaca fascicularis) is one of the most widely used surrogate animal models for an increasing number of human diseases and vaccines, especially immune-system-related ones. Towards a better understanding of the gene expression background upon its immunogenetics, we constructed a cDNA library from Epstein-Barr virus (EBV)-transformed B lymphocytes of a cynomolgus monkey and sequenced 10,000 randomly picked clones.ResultsAfter processing, 8,312 high-quality expressed sequence tags (ESTs) were generated and assembled into 3,728 unigenes. Annotations of these uniquely expressed transcripts demonstrated that out of the 2,524 open reading frame (ORF) positive unigenes (mitochondrial and ribosomal sequences were not included), 98.8% shared significant similarities (E-value less than 1e-10) with the NCBI nucleotide (nt) database, while only 67.7% (E-value less than 1e-5) did so with the NCBI non-redundant protein (nr) database. Further analysis revealed that 90.0% of the unigenes that shared no similarities to the nr database could be assigned to human chromosomes, in which 75 did not match significantly to any cynomolgus monkey and human ESTs. The mapping regions to known human genes on the human genome were described in detail. The protein family and domain analysis revealed that the first, second and fourth of the most abundantly expressed protein families were all assigned to immunoglobulin and major histocompatibility complex (MHC)-related proteins. The expression profiles of these genes were compared with that of homologous genes in human blood, lymph nodes and a RAMOS cell line, which demonstrated expression changes after transformation with EBV. The degree of sequence similarity of the MHC class I and II genes to the human reference sequences was evaluated. The results indicated that class I molecules showed weak amino acid identities (<90%), while class II showed slightly higher ones.ConclusionThese results indicated that the genes expressed in the cynomolgus monkey could be used to identify novel protein-coding genes and revise those incomplete or incorrect annotations in the human genome by comparative methods, since the old world monkeys and humans share high similarities at the molecular level, especially within coding regions. The identification of multiple genes involved in the immune response, their sequence variations to the human homologues, and their responses to EBV infection could provide useful information to improve our understanding of the cynomolgus monkey immune system.

Phytotoxicity on cotton ex-plants of an 18.5 kDa protein from culture filtrates of Verticillium dahliae

A phytotoxic protein that evokes the typical symptoms of Verticillium wilt disease in seedlings of Gossypium hirsutum L. (Upland cotton) was isolated from culture filtrates of Verticillium dahliae. The protein was purified by ammonium sulfate precipitation, Sephadex-G100 fractionation, and native PAGE. The 18.5 kDa protein, designated VD18.5, appears to be a single subunit protein with an isoelectric point between 3 and 5. VD18.5 induces symptoms of leaf dehydration, chlorosis, necrosis and stem discoloration in seedlings of the disease susceptible cotton cultivar Siokra 1–4. The LD 50 of VD18.5 on protoplasts of Siokra 1–4 was 18 μg mL −1. VD18.5 had no noticeable effect on Pima S-7, which is a disease resistant cultivar. Phytotoxic activity was partially destroyed at high temperature and was abolished by digestion with proteinase K. Mass spectrometry fingerprinting and protein sequence data from VD18.5 yielded no significant matches when submitted to the Mascot search engine and NCBI non-redundant protein databases, respectively. These results suggest that VD18.5 is a novel protein that may be involved in the development of some of the symptoms associated with Verticillium wilt disease in the cotton plant.